The present invention relates to methods for cryo-storage of specimens, in particular, for the production, storage, and manipulation of biological specimens in a cryo-preserved or in a thawed condition, such as, for example, a cryo-preservation method for biological cells. The invention relates also to methods for writing and reading of data. The invention further relates to a device for cryo-storage of specimens, in particular, a storage substrate for biological specimens, such as, for example, cells or cell components, a device for writing and reading of data in storage media and a cryo-bank system. The invention also relates to uses of the cryo-preservation of biological specimens.
Cryo-preservation is a commonly known method for maintaining in particular biologically or medically relevant materials. These materials include, for example, tissue and organs, body fluids, or also individual cells or cell components. The cryo-preservation takes place according to predetermined procedures in containers or on substrates, whose shape is adapted to the material or specimen. Containers for cryo-preservation are known, for example, for tissue and organs (see DE-OS 199 22 31, EP-A 0 853 238, DE-OS 197 25 768, DE-OS 199 05 163), for blood components (see, for example, DE-OS 198 26 350), and for cell or drop-shaped cryo-specimens (see, for example, U.S. Pat. No. 5,275,016, EP-B 0 475 409, DE-OS 199 21 236, EP-B 0 804 073).
A general concern with the cryo-preservation of biological specimens is in the ability of the specimen to be identified. Cryo-preservation specimens must be able to be identified with reference to their origin and characteristics with a high degree of certainty, without the necessity of thawing. With the macroscopic specimens, this is not a problem, since organ or blood containers can be provided with an inscription. Locating the cryo-specimen takes place in dependence on storage systems of the respective cryo-bank.
With small cryo-specimens in the form of frozen suspension drops, cells, cell aggregates, or cell components, the identification of the cryo-specimens is considerably problematic. A cryo-specimen would be negligibly small compared with the inscription. Often, an interest exists in the cryo-preservation of a plurality of microscopically small specimens. The storing and identification of small cryo-specimens with inscriptions would be impractical. In addition, the cryo-preserved cell specimens are available in a disordered state with common preservation techniques, which are based on the spraying of cell suspensions on cool surfaces (see, for example, EP-B 0 475 409). Only large amounts of individual specimens can be preserved commonly and unspecifically.
With the preservation techniques described in EP-B-8-4 073 and DE-OS 199 21 236, an arranged placement and specific processing of even the smallest specimens on the cryo-substrates is possible. The specimen deposition takes place, for example, with the use of a micro-drop shooting device, which is controlled on the basis of predetermined target coordinates. The specimens are located on defined substrate positions, on which also a specific measurement of specimen properties and identification of the specimens is possible. The substrate can be provided with a marking, in order to define the specimen positions on the substrate. For example, in DE-OS 199 21 236, for the matrix-type deposition of cryo-specimens in straight lines and columns, it is proposed that the substrate is provide with a designation of the columns and lines. This marking technique is illustrated in FIG. 27 (prior art).
The conventional marking of cry-substrates according to FIG. 27 has the following disadvantages. While the identification of specimens is possible, however, only information about the positions is provided. The limited information content of the substrate marking, however, represents a problem since, in addition to the specimen identification, also additional data, for example, about the condition or the history of the specimen or about measurement results should be available. These data can be stored in a parallel-operated data bank. The separate operation of cryo- and data banks, however, represents a considerable risk for the certainty of the feature allocation to the individual specimens. This risk is critical in particular with human medical uses, since a specific mistake can defeat the success of a further use of the cryo-specimen. In addition, the substrate marking has the disadvantage that specimen identification is possible only in connection with the cryo-substrate. When a specimen removal takes place, for example, such as that described in DE-OS 199 21 236, after separation from the cryo-substrate, a specimen identification can take place only by an expensive measurement of specific characteristics in a thawed state.
From DE-OS 197 52 085, a specimen carrier for microscopic analysis of a plurality of specimens is known. The conventional specimen carrier is formed as a substrate with a plurality of specimen receiving spaces, as shown in FIG. 28 in schematic plan view (prior art). The substrate, for example, has the shape of a plate storage medium (for example, a CD). Between a passage hole in the substrate center and the associated matrix-type specimen-receiving spaces, a ring region is formed. From DE-OS 197 52 085, it is known to form these ring regions for storing specimen data. The common specimen carrier has the disadvantage that it can only be used for receiving liquid specimens and not for cryo-preservation. In addition, the storage of specimen data on the inner ring represents the same disadvantage as the above-described substrate marking. More data can be stored; however, the association to the individual specimens is not possible without errors.
In addition to the noted disadvantages of the common technology, the following basis also exists for the minimally developed use, until now, of cryo-preservation, in particular, in cellular biotechnology. If a direct freezing of biological specimens takes place in a liquid cooling phase (for example, nitrogen), a risk of contamination exists. Over the cooling phase, viruses can be transmitted to the specimens. In order to avoid this risk, the contact with the liquid phase must be avoided or a sealed covering of the specimens must take place. Up to now, this has not been able to be realized in a practical manner.
From labor technology, specimen carriers, for example, in the form of object carriers or micro-titer plates are known, which are equipped with data storage media. These conventional specimen carriers are not suited for cryo-storage. First, they are merely suited for a use at ambient room temperature or a refrigerator temperature above the freezing point of water. A use at low temperatures has not been provided up to this point. Second, conventional specimen carriers are provided as substrates for specimens. Treatment, manipulation, or cultivation of the specimens takes place on the substrates. For storage in a preserved state, the conventional specimen carriers, however, are not suitable. In this connection, the containers for cryo-preservation are used, as described above. Finally, the conventional specimen carriers are not suitable for an effective specimen storage and manipulation. In practice, they must be manually transported; storage with high density is impossible.
The object of the present invention is to provide an improved method for cryo-preservation, with which the disadvantages of the conventional techniques are overcome, which has a broader range of use, and in particular, which is suited for automated preservation storage. The new method for cryo-preservation should make possible, in particular, that the specimen data are accommodated in greater amounts and with a higher data certainty (that is, with increased certainty of the association of specimen data to determined specimens). The invention also makes possible a highly specific data association to individual cryo-specimens. The object of the invention is also to provide devices for implementation of the improved cryo-preservation methods described above.